Project Summary Heart failure (HF) remains a significant cause of morbidity and mortality in the United States; each year, 1 million deaths and 250,000 hospitalizations are directly attributed to HF. Cardiac fibrosis is a driver of heart failure and valve disease. Clinically, cardiac fibrosis is a strong predictor of sudden cardiac death (SCD) in HF. Both endothelial-to-mesenchymal transition (EndMT) or epithelial-to-mesenchymal transition (EMT) are able to generate cardiac fibrosis in the adult heart which, in turn, predisposes individuals to HF, cardiac arrhythmias, and SCD. While many pathways have been found to contribute to cardiac fibrosis, inherited mutations causing fibrosis have not been readily identified. We have identified a family with a complex medical history characterized by extensive cardiac fibrosis and cardiac valve disease. We performed whole exome sequencing on affected members of this family and identified a mutation in muscle segment homeobox 1 (MSX1-E135D) that was present in all members of the family with disease. MSX1, also known as HOX7, is a transcription factor and a transcriptional repressor in the heart. MSX1 participates in EndMT during cardiac development, but remains poorly studied in the adult heart. The long-term objective of this application is to better understand the pathways and processes which underlie inherited cardiac fibrosis. The specific objective of this project is to understand the role of MSX1 in the adult heart and its contribution to cardiac fibrosis. We hypothesize that MSX1 regulates transcriptional repression of gap junctions and EndMT in the adult heart, and that the loss of this regulation leads to cardiac fibrosis in arrhythmias. We will test this hypothesis through two specific aims: 1) Define the physiologic role of Msx1 in the adult heart. We will characterize the effect of Msx1 cardiac- specific deletion in the adult mouse heart by electrocardiography, echocardiography, cardiac MRI (cMRI), invasive and non-invasive electrophysiology studies, and whole cell patch clamping of primary cardiomyocytes of Msx1 cardiac-specific knockout and littermate control mice. 2) Define the molecular pathways by which Msx1 deletion causes cardiac dysfunction. We will test the function of the MSX1-E135D mutation through luciferase assay of known genes repressed by MSX1. We will also test for enhanced fibrosis in the hearts of our knockout mouse by immunohistochemistry for makers of fibroblasts and endothelial cells. Also, we will use RNA- and ChIP-seq to define targets of Msx1 regulation in the heart. When successful, these studies will implicate a new transcription factor in the development of cardiac fibrosis, conduction disease, and HF. Further understanding of the function of MSX1 and its associated pathways in the adult heart may lead to new targets for therapeutic intervention in cardiomyopathic and arrhythmogenic conditions, supporting the Mission, Goals, and Objectives of the NHLBI.